Overall: Our project combines the significant advantages of a genetic model organism, sophisticated pathway mapping tools, high-throughput and accurate quantum chemistry (QM), and state-of-the-art experimental measurements. The result will be an efficient and cost-effective new approach for unknown compound identification in metabolomics, which is one of the major limitations facing this growing field of medical science. Caenorhabditis elegans has several advantages for this study, including over 10,000 available genetic mutants, well-developed CRISPR/Cas9 technology, and a panel of over 500 wild C. elegans isolates with complete genomes. Half of C. elegans genes have homologs to human disease genes, making this model organism an outstanding choice to improve our understanding of metabolic pathways in human disease. We will develop an automated pipeline for sample preparation to reproducibly measure tens of thousands of unknown features by UHPLC-MS/MS. We will use the wild isolates to conduct metabolome-wide genetic association studies (m- GWAS), and SEM-path to locate unknowns in pathways using partial correlations. The relevance of the unknown metabolites to specific pathways will be tested by measuring UHPLC-MS/MS data from genetic mutants of those pathways. Molecular formula and pathway information will be the inputs for automated quantum mechanical calculations of all possible structures, which will be used to accurately calculate NMR chemical shifts that will be matched to experimental data. The correct structures will be validated by comparing them with 2D NMR data of the same compound. The validated computed structures will then be used to improve QM-based MS/MS fragment prediction, using the experimental UHPLC-MS/MS data. The Administrative Core (AC) will be responsible for the coordination of all sites and scientific activities of the team. Prof. Edison, PD of the entire project, will direct the AC and will use his experience in managing large teams at different sites. He has done this with a number of other large projects spanning multiple research groups and universities. Edison has collaborated with every member of this team and has scientific background and interests covering most of the project. Our team members are all involved in large projects that are closely related to ours, and thus we will easily form new connections with the metabolomics community and beyond. Prof. Edison was the founding director of the Southeast Center for Integrated Metabolomics and thus has interacted extensively with all members of phase 1 of the NIH Common Fund Metabolomics Consortium. He also is a founding board member of the Metabolomics Association of North America, which will enable further connections to the community. Dr. Panagos will be our program manager and will coordinate regular interactions between scientific team members. The Administrative Core will prioritize pathways and unknowns for our study with the input of all team members and the NIH Consortium. We will establish and maintain a center website and database for our experimental and computational data.